Nucleic acids encoding trypanosoma cruzi heparin-binding protein

ABSTRACT

Disclosed is recombinant penetrin polypeptide. Also disclosed is nucleic acid encoding penetrin, recombinant cells and plasmids encoding penetrin, antibodies directed against penetrin and various uses for penetrin and antibodies directed against penetrin.

This invention was made with Government support under Grant No. AI18102awarded by the PHS. The Government has certain rights in the invention.

This is a divisional of copending application Ser. No. 07/779,706, filedOct. 17, 1993, issued Aug. 10, 1993 as U.S. Pat. No. 5,234,822.

BACKGROUND OF THE INVENTION

Trypanosoma cruzi, a parasitic protozoan, is the causative agent ofChagas' disease, a multisystemic disorder that affects millions ofpeople in Latin America. T. cruzi exists in three distinct developmentalforms: epimastigotes, which multiply extracellularly in the midgut ofreduviid bugs; amastigotes, which multiply inside mammalian cells; andtrypomastigotes, which transmit infection from insects to man andvice-versa, but which do not multiply

Trypomastigotes must travel through the bloodstream, cross the vascularepithelium, and migrate through the extracellular matrix to reach thecells of the organs which T. cruzi infects. Once they have entered hostcells, trypomastigotes transform into amastigotes which multiply andeventually transform back into trypomastigotes which can exit the hostcell and migrate through the interstitial tissue to invade other cells.

Receptor-ligand interactions may play a role in T. cruzi invasion ofcells. Ouaissi et al. (Science 234:603, 1986) report that peptidesmodeled on a protein, fibronectin, present on host cells can inhibit T.cruzi infection in vitro. Ouaissi et al. (Mol. and Biochem. Parasitol.19:201, 1986) report the identification and isolation 80-85 kDtrypomastigote cell surface protein with properties expected of afibronectin receptor. Velge et al. (Parasitology 97:255, 1988) reportthe isolation of a T. Cruzi trypomastigote collagen-binding protein.When analyzed by polyacrylamide gel electrophoresis, the proteinmigrates as a 58 kD protein under non-reducing conditions and as a 68 kDprotein under reducing conditions. Velge et al. also identify an 80-85kD protein which reacts with antibodies raised against the 68/58 kDprotein; they suggest that both the 80-85 kD and the 58/68 kDglycoproteins form part of the same receptor.

SUMMARY OF THE INVENTION

In general, the invention features recombinant penetrin polypeptide.Preferably, the polypeptide is derived from T. cruzi. Preferably suchpolypeptides have at least 70%, more preferably at least 80%, even morepreferably at least 90% homology to the penetrin of T. cruzi. By"derived from" is meant encoded by the genome of that organism.

The invention further features a substantially pure polypeptide which isa fragment or analog of penetrin capable of inhibiting T. cruziinfection of a eukaryotic cell. A T. cruzi infection inhibiting fragmentcan be identified using an assay described herein below. In this assayVero cells are exposed to T. cruzi in the presence and absence ofputative inhibitors and infection rates are measured. Portions ofpenetrin which are exposed on the surface of trypomastigotes are likelyto be better inhibitors than non-exposed portions.

The invention also features a substantially pure polypeptide which is afragment or analog of penetrin capable of promoting adhesion ofeukaryotic cells to a solid support surface.

By "penetrin polypeptide" is meant all or part of a trypomastigote cellsurface protein which promotes adhesion to and penetration of eukaryoticcells. By "polypeptide" is meant any chain of amino acids, regardless oflength or post-translational modification (e.g., glycosylation).Preferably a polypeptide includes at least 10, more preferably at least15, even more preferably at least 20, and even more preferably at least30 amino acids.

In a related aspect, the invention features an isolated nucleic acidwhich encodes a penetrin polypeptide (or fragment or analog thereof)described above. Preferably such nucleic acids have at least 70%, morepreferably at lest 80%, even more preferably at least 90% homology tothe penetrin encoding gene of T. cruzi. In a preferred embodiment, thenucleic acid includes a sequence substantially identical to the penetrinencoding portion of the DNA molecule identified as HBD-6, deposited withthe ATCC and given Accession No. ATCC 75126. By "substantiallyidentical" nucleic acid sequence is meant a sequence capable of encodinga substantially identical amino acid sequence.

In a related aspect, the invention features a plasmid which includes anisolated nucleic acid described above. In a preferred embodiment, theplasmid includes an expression control sequence capable of directingexpression of the penetrin polypeptide.

In a related aspect, the invention features a cell which includes anisolated nucleic acid described above. In various preferred embodiments,the cell is a prokaryotic cell; is a eukaryotic cell; and is a mammaliancell.

In another aspect, the invention features penetrin produced by a celldescribed above.

In another aspect, the invention features a purified antibody which iscapable of forming a specific immune complex with a penetrin polypeptide(or fragment or analog thereof). By "purified antibody" is meant onewhich is sufficiently free of other proteins, carbohydrates, and lipidswith which it is naturally associated to permit therapeuticadministration or use in ELISA-type assays of biological fluids.

In another aspect, the invention features a polypeptide which includesan amino acid sequence substantially identical to that encoded by thepenetrin coding portion of the DNA molecule identified as HBp-6deposited with the ATCC and given Accession No. ATCC-75126. By a"substantially identical" amino acid sequence is meant an amino acidsequence which differs only by conservative amino acid substitutions,for example, substitution of one amino acid for another of the sameclass (e.g., valine for glycine, arginine for lysine, etc.) or by one ormore non-conservative amino acid substitutions, deletions, or insertionslocated at positions of the amino acid sequence which do not destroy thebiological activity of the penetrin polypeptide.

In another aspect, the invention features a therapeutic compositionwhich includes, in a pharmaceutically acceptable carrier, asubstantially pure penetrin polypeptide (or fragment or analog thereof)as described above.

In another aspect, the invention features a method of detecting penetrinin a sample of biological fluid. The method includes: (a) contacting thesample of biological fluid with an antibody described above; and (b)detecting immune complexes formed in step (a), formation of immunecomplexes being an indication of the presence of T. cruzi penetrin inthe biological fluid.

In another aspect, the invention features a method of detecting anantibody reactive with penetrin in a sample of biological fluid. Themethod includes (a) contacting the sample of biological fluid with asubstantially pure penetrin polypeptide or an immunogenic fragmentthereof; and (b) detecting immune complexes formed in step (a),formation of the immune complexes being an indication of the presence ofthe antibody reactive with penetrin in the biological fluid.

In another aspect, the invention features a solid support surface forgrowing eukaryotic cells, the surface being coated with penetrin. Solidsupport surfaces for growing eukaryotic cells include: roller bottles,tissue culture plates, hollow fiber filter systems, and the like. Suchsurfaces can be coated with penetrin in much the same way as they arecoated with fibronectin.

In yet another aspect, the invention features a vaccine which includes asubstantially pure penetrin polypeptide or immunogenic fragment thereof.

By "isolated" is meant that the nucleic acid is largely free of thecoding sequences of those genes that, in the naturally occurring genomeof the organism from which the nucleic acid is derived, directly flankthe nucleic acid. Isolated nucleic acid may be genomic DNA, cDNA,chemically synthesized nucleic acid, enzymatically synthesized nucleicacid, or recombinant nucleic acid. The term includes chemically andenzymatically synthesized nucleic acid produced using a recombinantnucleic acid as a template. By "plasmid" is meant an extrachromosal DNAmolecule which includes sequences that permit replication within aparticular host cell. By "expression control sequence" is meant anucleotide sequence which includes recognition sequences for factorsthat control expression of a protein coding sequence to which it isoperably linked. Accordingly, an expression control sequence generallyincludes sequences for controlling both transcription and translation,for example, promoters, ribosome binding sites, repressor binding sites,and activator binding sites. "Homology" for amino acid sequences refersto the similarity between two or more amino acid sequences. The percenthomology of two given proteins is determined using sequence analysissoftware (e.g., the Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin, Madison, Wis.). Such softwaredetermines the homology of two amino acid sequences that have beenaligned so as to maximize homology. Homology values are assigned toexact matches as well as certain types of conservative amino acidsubstitutions. Such conservative substitutions include replacement ofone acidic amino acid by another acidic amino acido. For nucleic acidhomology is the percent identity between optimally aligned sequences.

By "substantially pure" is meant a polypeptide or protein which has beenseparated from components (e.g., other proteins) with which it isnormally found. Typically, a protein or polypeptide of interest issubstantially pure when at least 75% (preferably 85%) of the polypeptidein a sample is the protein or polypeptide of interest. By the term"capable of forming a specific immune complex" is meant an antibody doesnot substantially bind other molecules.

Besides substantially full-length penetrin, the present inventionprovides biologically active fragments of penetrin. As used herein, theterm "fragment", as applied to penetrin, will ordinarily be about 5contiguous amino acids, typically at least 10 contiguous amino acids,preferably at least 15 amino acids, and most preferably at least 20amino acids.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DETAILED DESCRIPTION

The drawings are first briefly described.

DRAWINGS

FIGS. 1A and B depict a pair of graphs illustrating the adherence of [³⁵S]-labeled trypomastigotes to various materials. In panel A,trypomastigote bound (cpm×10⁻³) to microtiter wells coated with 25 μg/mlheparin are plotted as a function of the incubation time (min). In panelB, trypomastigote bound (cpm ×10⁻³) to microtiter wells coated with 25μg/ml heparin are plotted as a function of the number of trypomastigote(×10⁻⁴) added to the microtiter well.

FIG. 2 is a graphical representation of the effect of the concentration(μg/ml) of heparin (filled squares), heparan sulfate (filled circles),collagen IV (open squares), hyaluronic acid (filled diamonds), orchondroitin sulfate (squares with central dot) bound to microtiter wellson binding of [³⁵ S]-labeled trypomastigote (cpm×10⁻⁴) bound.

FIGS. 3A and B depict a pair of graphs illustrating the adherence oftrypomastigotes to Vero cells. [³⁵ S]-labeled trypomastigote bound(cpm×10⁻⁴) are plotted as a function of incubation time (min) in panel Aand as a function of trypomastigote added (×10⁻⁵) in panel B.

FIG. 4 is a graphical representation of the effect of the concentration(μg/ml) of heparan sulfate (open circles), heparin (filled circles),chondroitin sulfate (open squares), or hyaluronic acid (filled squares)on inhibition of Vero cell infection (%).

FIG. 5 is a representation of an SDS-PAGE analysis of [¹²⁵ I]-labeledproteins in trypomastigote lysate (lane 1), 1.0M NaCl eluate (lane 2),and effluent from a heparin-Sepharose column (lane 3).

FIG. 6 is a representation of an immunoblot analysis of trypomastigote(lane 1) and amastigote (lane 2) lysates after 10% SDS-PAGE and transferto nitrocellulose using mouse anti-penetrin antibodies as a probe.Numbers at left indicate the position of markers having the indicatedmolecular weight (kD).

FIG. 7 is a graphical representation of the effect of severalproteoglycans on binding of [¹²⁵ I]-labeled column-purified penetrin toheparin-Sepharose;

FIG. 8 is a graphical representation of the effect of the concentration(μg/ml) of penetrin (open circles), recombinant penetrin (filledcircles), fibronectin (squares with central dot), BSA (x) used to coatpolystyrene wells on the adherence of Vero cells (cpm×10⁻³);

FIG. 9 is a graphical representation of the effect of the concentration(μg/ml) of penetrin (filled squares), heparin (open circles), orheparin-Sepharose depleted penetrin (open squares) on trypomastigoteinfection (% inhibition) of Vero cells.

T. CRUZI Adhesion Protein

The isolation and cloning of a novel T. cruzi adhesion protein,penetrin, is described below. In addition, experiments described belowdemonstrate that: penetrin binds selectively to proteoglycans andcollagen; penetrin promotes the adhesion and spreading of host cells onthe substratum; and that penetrin inhibits the ability of T. cruzitrypomastigotes to infect cultured cells. Other experiments demonstratethat when E. coli are made to express penetrin, they are able to adhereto and penetrate non-phagocytic fibroblasts.

T. cruzi

The Silvio X-10/4 clone of T. cruzi (Prioli et al., J. Immunol.144:4384, 1990) was used in all of the experiments described herein.

Trypomastigotes were obtained by infection of cultured Vero cells asdescribed by Prioli et al. (supra). After harvesting, the parasites werewashed three times in serum free RPMI-1640 and resuspended at thedesired concentration in phosphate-buffered saline (PBS, 150mM NaCl;20mM NaH₂ PO₄, pH 7.2) for radioiodination and competitive bindingassays, or in serum-free RPMI-1640 containing 1% BSA for adhesion andinfection experiments.

Adherence of T. cruzi Trypomastigotes to Immobilized Proteoglycans

Trypomastigotes adhered to heparin/heparan sulfate, collagen type I andcollagen type II that had been immobilized on a plastic surface. Incontrast, trypomastigotes bound poorly or not at all to chondroitinsulfate, hyaluronic acid, and the glycoproteins fetuin, asialofetuin,bovine submaxillary mucin, and hog gastric mucin.

For these adhesion experiments trypomastigotes were labeled with [³⁵ S]methionine as follows. Trypomastigotes (2×10⁸ cells/ml) werepreincubated in methionine-free RPMI 1640 containing 1% dialyzed-FCS.After 1h at 37° C., 250 mCi/ml of [³⁵ S] methionine (specific activity1113 Ci/mmole, ICN Biomedicals Inc., Irvine, Calif.) were added, andcontinued for 60 min at 37° C. The organism were washed three times inRPMI 1640 and adjusted to the appropriate parasite number in the samemedium. Incorporation of [³⁵ S] methionine into T. cruzi, determined byTCA precipitation, was usually about 50%, with a specific activityranging from 0.5I to 0.71 cpm/organism.

Proteoglycans (dissolved in PBS) and collagen (solubilized in 0.1N HCland neutralized in PBS) were adsorbed onto plastic (Falcon 3912polyvinylchloride 96-well microliter plates) by incubation overnight at4° C (Roberts et al., J. Biol. Chem. 264:9289, 1989). The unboundproteins were removed, the wells filled with 1% heat-treated BSA in PBS(56° C, 60 min and filtered in 0.22 mm nitrocellulose filters to removeinsoluble particles) and incubated for 1 h at room temperature. Thewells were then washed twice with PBS (pH 7.2) and rinsed with RPMI I640containing 1% BSA. T. cruzi trypomastigotes, labeled with [³⁵ S]methionine, were resuspended in RPMI-BSA and 100 μl of the suspensionwas applied to the wells. After various periods of incubation at 37° C.,the wells were carefully washed three times with 37° C. PBS (pH 7.2). T.cruzi bound to the plastic-bound proteoglycans were then solubilizedwith 100 μ l of 2 M NaOH, mixed with Ultrafluor (National Diagnostics,Manville, N.J.), and assayed by liquid scintillation counting. Referringto FIG. 1a, adherence of trypomastigotes to heparin-coated platesincreased linearly with the length of incubation up to about 60 min. Ina separate experiment trypomastigotes were added for 60 min, but thenumber added was varied. This experiment demonstrated that roughly20-40% of input trypomastigotes remained bound to the heparin coatedwells (FIG. 1B). In contrast, non-specific binding to BSA-coated wellswas less than 2%.

Referring to FIG. 2, heparin (filled squares) heparan sulfate (filledcircles), and collagen IV (open squares) were equally effective inpromoting trypomastigote adherence. Chondroitin sulfate (squares withcentral dot) and hyaluronic acid (filled diamonds) did not promoteadhesion. A similar set of experiments demonstrated that collagen I, butnot fetuin, asialofetuin, bovine submaxillary mucin, or hog gastricmucin promote adhesion. Soluble heparin, soluble heparan sulfate andsoluble collagen inhibited trypomastigote binding to heparin- orcollagen-coated wells. Proteoglycans Neutralize T. cruzi

Adherence to Vero Cells

The adherence of T. cruzi to glutaraldehyde-fixed fibroblasts (Verocells) was found to be dose dependent, time dependent and saturable. Lowconcentrations of heparin or heparan sulfate inhibited adhesion oftrypomastigotes to Vero cells. In contrast, incubation with chondroitinsulfate, hyaluronic acid, bovine submaxillary mucin, hog gastric mucin,fetuin, or asialofetuin did not affect adhesion. Preincubation of T.cruzi with heparin or heparan sulfate followed by washing to removeunbound proteoglycans blocked adhesion. Similar treatment of Ver cellsprior to incubation with T. cruzi had no effect on adhesion.

In these experiments attachment of trypomastigotes to Vero cellmonolayers was measured as follows. Briefly, Vero cells were maintainedin RPMI 1640 supplemented with heat-inactivated 1% Nu-serum(Collaborative Research Inc., Bedford, Mass.), penicillin (100 U/ml) andstreptomycin (100 mg/ml). Unless noted otherwise, cells were maintainedin plastic tissue culture flasks (150 cm², Bellco, Vineland, N.J.) andincubated at 37° C. in a humidified atmosphere containing 5% CO₂ and 95%air. Cells were propagated every 1-2 weeks after trypsin-EDTA digestion(0.5 g trypsin, 0.2 g EDTA in HBSS without Ca⁺⁺ and Mg⁺⁺) in 16-wellLab-Tek® chamber slide™ (Nunc, Inc., Naperville, Ill. Prior to bindingexperiments Vero cells were removed with trypsin and plated on 96-welltissue culture plates (Bellco, Vineland, N.J.) or in 16-well Lab-Tek®chamber slide™ at a density of 7.5×10³ cells per well. After 24 h, themedium was removed, cells were washed twice with PBS, pH 7.2, and fixedfor 5 min at 4° C. by addition of 2% glutaraldehyde (grade I, SigmaChemical Co., St. Louis, Mo.) in PBS. Cell monolayers were washed twicewith PBS, pH 7.2 and incubated overnight at 4° C. in 0.16M ethanolamine,pH 8.3 to block residual free amino groups. The next day, cellmonolayers were washed three times with PBS as above, and two times with1% BSA/RPMI-1640 and kept at least 1 h at 37° C. in the same medium.

Trypomastigotes, [³⁵ S]-labeled as described above, were added to thecell monolayers in a total volume of 100 μl well and the plates wereincubated for 1 h at 37° C. Each well was then washed five times with100 μl prewarmed PBS (pH 7.2). The [³⁵ S] label was solubilized with 2MNaOH and the bound radioactivity was determined in a liquidscintillation counter. Alternatively, unlabeled trypomastigotes wereadded to wells of a 16-well Lab-Tek® chamber slide™ and incubated asabove. After removing the plastic chamber, the slide was washed bydipping 5 times in PBS (pH 7.2) and immersed upside-down in 4%paraformaldehyde in PBS, containing 7% sucrose. Finally, the slide wasrinsed with PBS, stained with Diff-Quik® and the number of bound T.cruzi were determined by counting under light microscopy. For inhibitionstudies, trypomastigotes were preincubated 30 min at 37° C. with theappropriate proteoglycan in 100 μl of 1 % BSA-RPMI-1640 before additionto cell monolayers. Binding was determined in triplicate at eachinhibitor concentration and in the absence of inhibitor.

Referring to FIGS. 3A and 3B, binding studies demonstrated thattrypomastigote adherence to Vero cells was dependent on incubation timeand the number of T. cruzi added. Binding was also shown to besaturable. In these experiments 100% binding was taken as 5×10⁵trypomastigotes/well for 60 min.

Table 1 presents the results of the binding inhibition assay. In thisassay 100% binding was taken to 5 x10⁴ trypomastigotes/well bound in 60min.

                  TABLE 1                                                         ______________________________________                                        Inhibition of Trypomastigote Binding to Vero Cells                                                      % Vero Cells with                                   Inhibitor     Conc. (μg/ml)                                                                          Trypomastigotes                                     ______________________________________                                        BSA           250         95                                                  Heparin       125         50                                                                250         12.5                                                Heparan Sulfate                                                                             125         60                                                                250         18                                                  Hyaluronic Acid                                                                             250         98                                                  Chonodroitin Sulfate                                                                        250         96                                                  Bovine Submaxillary                                                                         250         96                                                  Mucin                                                                         ______________________________________                                    

These results are consistent with the existence of a heparin-bindingligand(s) on T. cruzi that mediates adherence by recognizingheparin-like receptor(s) on the surface of fibroblasts.

Inhibition of T. cruzi Infection by Heparin and Heparan Sulfate

Both soluble heparin and heparan sulfate inhibited infection of Verocells by trypomastigotes whereas chondroitin sulfate and hyaluronic acidhad little effect on infection. Incubation of Vero cells with heparin orheparan sulfate followed by washing had no effect on infection.

In these experiments, cultured Vero cells were released withtrypsin/EDTA, plated at 7.5×10³ cells/well in 16-well Lab-Tek® chamberslides™ for 24 h, and used for infection at ˜75% confluence.Trypomastigotes were harvested, washed with serum-free RPMI containing1% BSA, and mixed with various concentrations of proteoglycans. After 30min at 37° C., 5×10⁵ parasites in a total volume of 100 μl were added toeach well containing subconfluent cultures of Vero cells and allowed tointeract for 2 h at 37° C. At the end of the incubation time, cellmonolayers were rinsed three times with serum-free medium, and furtherincubated in RPMI-1640 containing 1% Nu-serum for 3 days at 37° C.Finally, monolayers were washed, stained with Diff-Quik®, and examinedunder a microscope to estimate the ratio of infected (i.e., Vero cellcontaining more than one intracellular amastigote) to non-infected Verocells. In order to establish if the inhibitory effect was due tointeraction of heparin and heparan sulfate with trypomastigotes ratherthan to Vero cells, parasites or host cells were preincubated with theputative inhibitors, washed extensively, and assayed for infection asabove.

Referring to FIG. 4, heparan sulfate (open circles) was found to be apotent inhibitor of infection, causing a 50% reduction at 1.5 μg/ml.Heparin (filled circles) reduced infection by 50% at 100 μg/ml.Chondroitin sulfate (open squares) and hyaluronic acid (filled squares)had little effect on infection even at high concentrations.

Isolation and Identification of Penetrin

Because it appeared that the protein mediating trypomastigote binding tofibroblasts is a heparin binding protein, a heparin-Sepharose column wasused in an attempt to purify the T. cruzi factor.

The trypomastigotes were first [¹²⁵ I]-labeled Tissue culturetrypomastigotes were washed three times by centrifugation as above andtheir surface proteins labeled by Iodo-gen™(1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril) catalyzed iodination asdescribed (Howard et al., J. Protozool. 29:114, 1982; Markwell, Anal.Biochem. 125:427, 1982). Briefly, washed parasites were resuspended at10⁸ cells/ml in cold PBS and added to glass vials coated with Iodo-gen™.Then, 500 μCi of carrier-free [¹²⁵ I] were added and the suspensionincubated 15 min on ice with intermittent agitation. To stop thereaction, the parasite suspension was transferred to a 15 ml conicaltube and washed three times in cold PBS, pH 7.2. Alternatively,parasites were labeled with Iodo-beads™ (N-chloro-benzenosulfomidesodium salt, Pierce Chemical Co., Rockford, Ill., using 4 beads (1.2μmoles) and 500 μCi of carrier-free [¹²⁵ I]. After 15 min on ice,parasites were removed and washed as above.

For purification of the heparin binding protein, [125I]-surface labeledtrypomastigotes (2.5×10⁸) were lysed by sonication, unbroken cellsnuclei and debris were removed by low speed centrifugation (100×g, 5min) and membranes were isolated by ultracentrifugation (100,000×g, 1h),washed twice with PBS and then lysed in a solution containing 1% TritonX-100 and an anti-protease cocktail (10mM pepstatin, 10mM leupeptin,10mM iodoacetamide, 10 μg/ml soybean trypsin inhibitor, and 2mM PMSF)for 1 h on ice. Soluble membrane proteins were isolated bycentrifugation at 15,000×g for 30 min at 4° C. and immediately mixedwith an equal volume of heparin-Sepharose. After overnight incubation at4° C. with gentle shaking, heparin-Sepharose, along with bound material,was removed by centrifugation (175×g for 10 min; at 4° C.), resuspendedin PBS, poured into a column (0.8×4 cm) and washed with 0.05% TritonX-100 in PBS, pH 7.2 until radioactivity in the effluent Was backgroundlevels (i.e., <100 cpm/100 μl ). Finally, bound proteins were eluted bystepwise addition of 25 ml of 0.25M, 0.5M, 1.0M and 3.0M NaCl, 1.5Mguanidine-HCl and 2.0 M potassium thiocyanate; for each addition, 2-mlfractions were collected. The radioactivity of the eluted fractions wasmonitored on a gamma counter, and fractions corresponding to theradioactive peaks pooled, dialyzed extensively against 200mM ammoniumbicarbonate and lyophilized. A single sharp peak of [¹²⁵ I]-labeledprotein was eluted by 1.0M NaCl. Further washing with 3.0M NaCl, 1.5Mguanidine-HCl and 2.0M potassium thiocyanate did not elute further [¹²⁵I]-labeled material. Based on the radioactivity applied to the column(75% of which was TCA precipitable) and radioactivity recovered in the1.0M NaCl peak, the eluted material represents ˜4.1% of labeled surfaceproteins.

Analysis of the 1.0M NaCl eluate by SDS-PAGE and autoradiographyrevealed a single 60 kD band (FIG. 5, lane 2), whose mobility in thepolyacrylamide gel was not changed by reduction with 2%2-mercaptoethanol. A 60 kD heparin-binding protein was similarlyisolated from trypomastigotes that had been endogenously labeled with ³⁵S]-methionine. An additional band of 32 kD was inconsistently observedin the 1.0M NaCl eluate and its absence or relative abundance was notaltered by reducing agents. Also, antibody to the 60 kD proteinsometimes recognized a 32 kD band in trypomastigote lysates.Furthermore, when the 32 kD band was present in the 1M NaCl eluate, itclearly bound heparin as judged by various binding assays. Therefore,the 32 kD band appears to be a degradation product of the 60 kDpolypeptide.

Non-infective epimastigotes, unlike infective trypomastigotes, did notadhere to plastic surfaces coated with proteoglycans. Consistent withthis observation, the 60 kD heparin-binding protein found intrypomastigotes was not detectable in epimastigotes. Furthermore,antibodies raised against the 60 kD protein (described below) did notreact with amastigote lysates by immunoblot (FIG. 6, lane 2). The 60 kDsurface protein is therefore developmentally regulated. It was namedpenetrin, on the basis of the experiments described below.

Penetrin Binds to Fibroblasts by a Receptor-Ligand Interaction

To determine if [¹²⁵ I]-labeled penetrin was still active in bindingextracellular matrix proteins after isolation by heparin affinitychromatography, rebinding and inhibition of rebinding toheparin-Sepharose beads was performed. Briefly, column purified [¹²⁵I]-labeled penetrin was incubated with or without compounds and mixedwith 100 μl of heparin-Sepharose. The mixture was incubated at roomtemperature for 2 h under slow rocking. The slurry was then centrifuged(500×g, 5 min), washed three times with PBS, and bound radioactivitydetermined in a gamma counter. Referring to FIG. 7, in the absence ofcompetitors, column-purified [¹²⁵ I]-labeled penetrin retained itsability to bind heparin-Sepharose.

For inhibition experiments, an aliquot of the purified heparin-bindingprotein was mixed with putative inhibitors for 1 h at room temperature,and the mixture added to 100 μl of heparin-Sepharose as above. Theamount of [¹²⁵ I]-labeled protein retained by the agarose beads wasassessed by elution of the protein with 1% SDS, SDS-polyacrylamide gelelectrophoresis and autoradiography, and quantitated in a laser scanningdensitometer (Pharmacia LKB, Ultroscan XL). One hundred percent bindingwas taken as the amount of protein retained by the heparin-Sepharose inthe absence of added inhibitor. Referring to Table 2, adsorption ofpenetrin was inhibited by pre-incubation with soluble heparin, heparansulfate or collagen, but not with other glycosaminoglycans (chondroitinsulfate and hyaluronic acid). Glycoproteins such as bovine submaxillarymucin, hog gastric mucin, and fetuin did not affect penetrin binding.Consistent with the conclusions derived from the binding of viableparasites to proteoglycans (FIG. 1), re-binding of penetrin toheparin-Sepharose was also inhibited by collagen, which was as good aninhibitor as heparin and heparan sulfate.

                  TABLE 2                                                         ______________________________________                                        Effect of Various Compounds on Binding of Column-Purified                     Penetrin to Heparin-Sepharose                                                 Compound (100 μg/ml)                                                                       Penetrin Bound (cpm × 10.sup.-3)                        ______________________________________                                        Bovine Serum Albumin                                                                          1.9                                                           Heparin         0.7                                                           Heparan Sulfate 0.75                                                          Chonodroitin Sulfate                                                                          1.6                                                           Hyaluronic Acid 1.8                                                           Collagen        0.6                                                           ______________________________________                                    

Penetrin was biologically active, as it bound to subconfluent monolayersof Vero cells. Binding of the [¹²⁵ I]-labeled protein to Vero cellmonolayers was dose dependent and saturable, and it was inhibited byheparin, heparan sulfate and collagen, but not by chondroitin sulfateand hyaluronic acid. The inhibition pattern was similar to that in theabove-described heparin-Sepharose was used as a binding probe, or to theprofile of trypomastigote binding to matrices coated with heparin andother extracellular matrix proteins. Therefore, the binding specificityof the isolated penetrin reproduces the binding characteristics ofintact trypomastigotes.

Binding of [¹²⁵ I]-labeled penetrin to Vero cells was assayed asfollows. Vero cells, grown in multiwell plates to confluence asdescribed above, were rinsed once with 1% BSA-RPMI-1640, and incubatedat 37° C. with increasing concentrations of [¹²⁵ I]-labeled penetrin.After 1 h the medium was removed, cell monolayers were washed threetimes with PBS, solubilized with 100 μl of 2M NaOH, and the retainedradioactivity measured in a gamma counter. For inhibition experiments,an aliquot of [¹²⁵ I]-labeled penetrin was preincubated with thepotential inhibitors prior to adding to confluent cells, and processedas above.

Referring to Table 3, heparin, collagen, and heparan sulfate, but notchondroitin sulfate inhibited binding of [¹²⁵ I]-labeled penetrin tofibroblasts.

                  TABLE 3                                                         ______________________________________                                        Effect of Various Compounds on Binding of                                     Column-Purified Penetrin to Fibroblasts                                       Compound     Concentration (μg/ml)                                                                      % Inhibition                                     ______________________________________                                        Heparin      0               0                                                             5               25                                                            25              55                                                            50              75                                                            100             89                                                            250             91                                               Collagen     100             60                                                            250             95                                               Heparan Sulfate                                                                            250             65                                               Chondroitin Sulfate                                                                        250             12                                               ______________________________________                                    

Cloning of Penetrin

A T. cruzi trypomastigote (Silvio X-10/4) genomic library in λZAP wasscreened with antisera monospecific for penetrin. Antisera were producedin C3H mice after three or more injections (10 days apart), inincomplete Freund's adjuvant, of affinity purified penetrin, or of the60 kD penetrin band cut-out from the polyacrylamide gel after SDS-PAGE.The penetrin antibodies reacted with a 60 kD polypeptide in lysates oftrypomastigotes, as determined by immunoblots and immunoprecipitation of[³⁵ S]-methionine labeled parasites. The antibody alsoimmunoprecipitated penetrin labeled with ¹²⁵ I after surface iodinationof trypomastigotes with Iodobeads. Ten DNA clones in E. coli XI-1 Blue(Short et al., Nucl. Acids. Res. 16:7583, 1988) were identified withthis antibody by standard techniques. Bacterial lysates, after SDS-PAGEand transfer to nitrocellulose, revealed a band of 62 kD recognized bythe penetrin antibody. The size of recombinant penetrin was thereforeclose to endogenous penetrin (60 kD). Recombinant penetrin, like itsendogenous counterpart, is a heparin-binding protein, as it was retainedby heparin-Sepharose columns and was eluted at 1M NaCl.

Adherence of Fibroblasts to Endogenous and Recombinant Penetrin

If penetrin binds to a specific receptor(s) on the surface of mammaliancells, then it is possible that the binding will promote cell adherenceto the substratum, analogous to the property of some mammalian adhesiveproteins, including fibronectin and vitronectin. This possibility wastested by determining if polystyrene wells that had been coated withpenetrin would provide a substratum for Vero cell adhesion andspreading.

Various concentrations of endogenous and recombinant penetrin, or offibronectin, all diluted in PBS (pH 7.2), were used to coat 96-wellmicrotiter plates by incubation overnight at 4° C. After washing withPBS, wells were blocked with 1% heated BSA (60 min at 56° C.) in PBS for2 h at room temperature, washed two times with PBS and incubated at 37°C. with 1% BSA, RPMI 1640 medium until needed. To assay for cellattachment, Vero cells were dispersed from confluent monolayers inHanks' balanced salt solution (Gibco, 310-4180AG), containing 10mM EDTA,washed three times with RPMI 1640 medium and resuspended inmethionine-free medium. After 30 min at 37° C., 50 μCi/ml [³⁵S]-methionine were added (New England Nuclear, Boston, Mass.) andincubated for 1 h at 37° C. Cells were washed thrice in RPMI 1640 mediumand resuspended at 10⁶ cells/ml in RPMI 1640 medium containing 1% BSA.Aliquots of [³⁵ S]-methionine-labeled Vero cells were added to each welland incubated at 37° C. After appropriate incubation times, the wellswere washed by gently aspirating the culture medium, and adding 100 μlwarm RPMI 1640. Cells that remained attached after two washes weresolubilized by addition of 100 μl of 2N NaOH and counted by liquidscintillation. In selected assays, attached cells were visualized afterfixation with 4% paraformaldehyde and staining with Diff-Quik. Theresults showed that after adhering to matrices coated with penetrin,Vero cells lost their round form and spread, as did the cells infibronectin-coated wells. In contrast, no adherence and spreading ofcells occurred in the wells coated with BSA.

Referring to FIG. 8, Vero cell adherence to penetrin (open circles) wasdose-dependent, and interestingly, more efficient than adherence tofibronectin (squares with central dot) at concentrations higher than 20μg/ml. Attachment of fibroblasts to penetrin was confirmed by usingpolystyrene wells coated with recombinant penetrin (filled circles)expressed in Escherichia coli. Recombinant penetrin, like its endogenouscounterpart, promoted attachment of fibroblast to the substratum andspreading, although it was somewhat less effective than endogenouspenetrin. BSA (x) did not promote adherence.

Inhibition of T. cruzi Infection by Penetrin

That penetrin mediates T. cruzi invasion of host cells was demonstratedby a series of experiments showing that soluble endogenous penetrininhibits infection. For this experiment subconfluent monolayers of Verocells were preincubated with penetrin or heparin for 30 min followed byincubation with trypomastigotes (5×10⁵) for 3 h in the presence ofvarious concentrations of penetrin, washed with RPMI to remove unboundparasites (and penetrin), and incubated for 3 d at 37° C. to allow T.cruzi to differentiate and replicate inside the host Vero cells.Monolayers were washed, stained with Diff-Quik®, and examined under amicroscope to estimate the ratio of infected (i.e., Vero cellscontaining more than one intracellular amastigote) to non-infected Verocells.

Referring to FIG. 9, T. cruzi infection was effectively blocked bypenetrin (filled squares) in a saturable manner, with concentration aslow as 3 μg/ml producing a detectable degree of inhibition. Thedose-response curve produced by the T. cruzi protein was comparable tothat of the penetrin-binding proteoglycan, heparin (open circles). Theeffect of penetrin on infection was completely abrogated by adsorptionof the protein to heparin-Sepharose (open squares) in agreement with theproperty of penetrin to bind heparin. This adsorption was accomplishedby applying penetrin at the indicated concentration to aheparin-Sepharose column and concentrating the effluent.

Penetrin Mediated Entry of E. coli into Fibroblasts

E. coli expressing penetrin are able to enter non-phagocytic mammaliancells which normally exclude E. coIi. This was demonstrated using E.coli XL-1 Blue transferred with clone HBP-6, that expresses penetrin;clone 7F, that does not express penetrin but does express T. cruzineuraminidase; or Bluescript. Recombinant bacteria were grown overnightat 37° , washed twice in PBS, resuspended at 10⁸ cells/ml, and aliquotsof 2×10⁷ E. coli were added to monolayers of 2×10⁵ Vero cells permicroliter well in 1 ml of RPMI 1640 medium containing 1% BSA. Bacteriawere centrifuged onto the cell monolayer, and the cultures wereincubated at 37° for 3 h in a 5% CO₂ /95% air atmosphere, washed threetimes with sterile PBS, and incubated for an additional 1.5 h at 37° C.in RPMI 1640 medium containing 1% BSA and 75 μg/ml gentamicin (to killbacteria bound to outer membrane). The monolayers were then washed threetimes in sterile PBS, and lysed with 0.5% Triton X-100 in deionizedwater to release internalized bacteria, which were quantitated in agarplates. For inhibition of bacterial entry, E. coli cells werepreincubated with 250 μg/ml of the putative inhibitors in 1 ml of RPMI1640, 1% BSA, added to each well of the monolayer and invasionquantitated by gentamicin-resistant viable bacteria as above.

The clone expressing penetrin was very effective in entering E. coli,whereas E. coli expressing the T. cruzi neuraminidase or transformedwith pBluescript were not. An average of 4.3% of the penetrin-expressingE. coli that were added to the fibroblast monolayer survived theantibiotic treatment. Further, the same extracellular matrix proteinsthat reacted with intact T. cruzi and with isolated penetrin were alsoeffective in preventing E. coli entry into Vero cells. Thus, collagentype IV at 250 μg/ml completely abrogated penetration of the bacteria inmammalian cells (<0.001% survival) and heparin and heparan sulfate, atthe same concentration, inhibited penetration by 85% (0.6% survival).Chondroitin sulfate and hyaluronic acid were without effect in theassay. E. coli transformed with the T. cruzi neuramidase gene orXL1-Blue alone could not

The ability of E. coli HBP-6 to penetrate Vero cells was confirmed bytransmission electron microscopy. The invasion assay was performed asdescribed above, except that 8×10⁷ bacteria were added to 8×10⁵ Verocells in tissue culture wells (Falcon, 6-well plates). The samples werethen fixed, embedded, sectioned and stained as described by Isberg etal. (Nature 317:262, 1985). Infected monolayers were fixed with 2%glutaraldehyde and 2% osmium tetroxide in 0.1M cacodylate buffer (pH7.4) and stained with uranyl acetate. Samples were dehydrated withethanol, embedded in Epon, thin sectioned, and poststained with uranylacetate and lead acetate.

Microscopy of Vero cells exposed to E. coli transformed with clone HBP-6revealed bacteria in direct contact with the outer membrane offibroblasts or intracellularly. In contrast, no bacteria were foundinside Vero cells exposed to E. coli transformed with clone 7F. Mostintracellular bacteria were not surrounded by host cell membranes, butinstead were in direct contact with the cytosol.

To determine if HBP-6 bacteria would replicate inside the Vero cells,monolayers were incubated at 37° C. for an additional 18 h in thepresence of gentamicin, and surviving bacteria were quantitated eitherby the agar plate technique or electron microscopy. No evidence ofintracellular multiplication of the bacteria was noted. The presence ofintracellular bacteria did not produce any obvious morphologicalalteration in the infected culture cells. Likewise, the intracellularbacteria were morphologically normal and alive, as judged by theirability to form colonies on agar after being released from thefibroblasts by detergent lysis. In addition, if thegentamicin-containing medium was replaced with antibiotic-free medium,E. coli HBP-6 grew abundantly in the culture supernatant after overnightincubation at 37° C., whereas similar antibiotic replacement did notproduce bacterial growth in the monolayers that had been exposed to E.coli transformed with clone 7F or with E. coli transformed withpBluescript. These results suggest that intracellular bacteria capableof expressing penetrin can be released into the medium.

If penetration of Vero cells by E. coli transformed with clone HBP-6 ismediated by penetrin, then the recombinant T. cruzi protein should beexposed on the surface of the bacteria. This prediction was confirmed byindirect immunofluorescence (Leong et al., EMBO J. 9:1979, 1990), whichrevealed a bright fluorescence of intact, viable bacteria, produced byanti-penetrin antibodies. Penetrin antibodies did not react with E. colitransformed with clone 7F, nor did normal sera bind to intact E. colitransformed with clone HBP-6. It is of interest to note that only about10% of HBP-6 bacteria were recognized by the penetrin antibodies, closeto the proportion of bacteria (4.3%) that invaded the Vero cellmonolayer. This finding provides further support for the argument thatthe invasive bacterial phenotype is represented by the bacterialsubpopulation recognized by the penetrin antibodies.

For the experiments described above, heparin, heparan sulfate,chondroitin sulfate, hyaluronic acid, collagens type I and IV, bovineserum albumin, cyanogen bromide, Sepharose® CL-4B-200, andphenylmethyl-sulfonyl fluoride (PMSF) were purchased from Sigma ChemicalCo., St. Louis, Mo. Iodine¹²⁵ (892 mCi/ml) was from ICN Radiochemicals,Irvine, Calif. Iodo-gen™ and Iodo-beads™ were obtained from PierceChemical Co., Rockford, Ill. All culture reagents were obtained fromGibco Laboratories (Grand Island, N.Y.). Heparin was coupled to cyanogenbromide-activated Sepharose® CL-4B as described (March et al., Anal.Biochem. 60:149, 1974).

Antibodies

Antibodies directed against penetrin can be used to diagnostically todetect T. cruzi in a sample of biological fluid. Such antibodies can beproduced using intact penetrin or immunogenic fragments thereof. Theportion of penetrin whose DNA and amino acid sequences are presented inSEQ ID NO: 1 and SEQ ID NO: 2 respectively may be useful for preparationof antibodies. Because this portion of penetrin is adjacent to ahydrophobic region and is relatively hydrophilic, it may be exposed onthe surface of T. cruzi The deduced amino acid sequence of penetrin canbe analyzed to locate regions that are likely to be immunogenic usingmethods described in Current Protocols in Immunology (Coligan et al.(eds.) Greene Publishing Associates and John Wiley & Sons, Inc., NewYork, 1991). Antibodies can be produced and used in an ELISA assay orother standard assay format according to standard procedures well knownto those in the art (Current Protocols in Immunology, Coligan et al.(eds.) Greene Publishing Associates and John Wiley & Sons, Inc., NewYork, 1991).

Penetrin, or immunogenic fragments thereof, can be used in variousimmunological assays to detect the presence of anti-penetrin antibodiesin the bodily fluids of patients suspected of being infected with T.cruzi. Methods for detecting antibodies using a protein or fragmentthereof are described in Current Protocols in Immunology (Coligan et al.(eds.) Greene Publishing Associates and John Wiley & Sons, Inc., NewYork, 1991).

Therapy

Since penetrin binds to heparin proteoglycans such as, heparan sulfate,heparin, and derivatives of heparin lacking anti-clotting properties,can be used to interfere with trypomastigote infection of cells within ahuman patient. Penetrin itself, or infection inhibiting fragmentsthereof, may be used to interfere with trypomastigote infection of cellswithin a human patient.

The following infection inhibition assay can be used to determine if aparticular penetrin fragment, or heparin derivative, inhibits T. cruziinvasion of host cells. Briefly, subconfluent monolayers of Vero cellsare preincubated with the candidate compound for 30 min followed byincubation with trypomastigotes (5×10⁵) for 3 h in the presence ofvarious concentrations of the candidate compound, washed With RPMI toremove unbound parasites (and candidate compound), and incubated for 3 dat 37° C. to allow T. cruzi to differentiate and replicate inside thehost Vero cells. Monolayers are washed, stained with Diff-Quik®, andexamined under a microscope to estimate the ratio of infected (i.e.,Vero cell containing more than one intracellular amastigote) tonon-infected Vero cells.

These compounds can be administered in an effective amount either aloneor in combination with a pharmaceutically acceptable carrier or diluent.All of the suitable compounds or compositions can be administered aloneor in combination with other therapeutic agents. They may beadministered by any convenient means, e.g., intravenously,intramuscularly, subcutaneously or intranasally. The compounds can beeffective whether administered during the acute of chronic phase ofinfection. Dosage will generally be in the range of 1 to 100 μg/ml perkilogram of body weight.

Penetrin, preferably recombinant, or immunogenic fragments thereof maybe used in a vaccine composition to vaccinate those at risk for becominginfected with T. cruzi Vaccine compositions may include penetrin (orfragments thereof) stabilizers, antibiotics and a pharmaceuticallycarrier.

Use

Penetrin can be used to coat solid support surfaces (e.g., tissueculture plates or hollow fibers) for growing eukaryotic to provide asurface that promotes attachment and spreading of the eukaryotic cells.

Deposit

Plasmid HBP-6 has been deposited with the American Type CultureCollection on Oct. 17, 1991 and bears the accession number ATCC No.75126. Applicants' assignee, New England Medical center Hospitals, Inc.,acknowledges its responsibility to replace this deposit should thisdeposit die before the end of the term of a patent issued hereon, andits responsibility to notify the ATCC of the issuance of such a patent,at which time the deposit will be made available to the public. Prior tothat time the deposit will be made available to the Commissioner ofPatents under the terms of 37 CFR Section 1.14 and 35 USC Section 112.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 394                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GCTGCACATCGTCGTCC ACCGGCAACACATTTAGTGATGCGCTGC45                              TCAGCCCGGAGGTGTGTTTCCACCGCACACGAACATCACGATCAG90                               TGGGAACCGCTTCACGGTCACGAGGCCGATCCCTCGGTCGGGTTT135                              GGTCCTTTGGGGCCCG TCGTGTGTCGCGATGAATGGACTGGCGAT180                             CAGCAACGACTCCGCAGTGGTGCTGAGTGGCAATGTGTTTCACGC225                              CGTGGCGCATCGTCAAGCGTCATTTACTTTGTTGGATTTGCGCTG270                              AGGGTGTCGTGGCAC TCCGTGTTTGCGGTGATGGGCAACACGTTT315                             CATGTGGCTGGCGCTAACAGTAGCGCTATAACTCTTGAGGGGTCT360                              AGCAGTACTTTACCTGATTGTGCTGACACTCGTC394                                         (2) INFORMATION FOR SEQ ID NO: 2:                                             ( i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 131                                                               (B) TYPE: amino acid                                                          (D) TOPOLOGY: Not Relevant                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      AlaAlaHisArgArgProProAlaThrHisLeuValMetArgCys                                 51015                                                                          SerAlaArgArgCysValSerThrAlaHisGluHisHisAspGln                                202530                                                                        TrpGluProLeuHisGlyHisGluAlaAspProSerValGlyPhe                                  354045                                                                       GlyProLeuGlyProValValCysArgAspGluTrpThrGlyAsp                                 505560                                                                        GlnGlnArgLeuArgSerGlyAla GluTrpGlnCysValSerArg                                657075                                                                        ArgGlyAlaSerSerSerValIleTyrPheValGlyPheAlaLeu                                 8085 90                                                                       ArgValSerTrpHisSerValPheAlaValMetGlyAsnThrPhe                                 95100105                                                                      HisValAlaGlyAlaAsnSerSerAlaIleThrLeuGluGlySer                                  110115120                                                                    SerSerThrLeuProAspCysAlaAspThrArg                                             125130                                                                    

We claim:
 1. A substantially pure nucleic acid consisting of anucleotide sequence set forth as SEQ ID NO:
 1. 2. A substantially purenucleic acid consisting of a nucleotide sequence that encodes theprotein set forth as SEQ ID NO:
 2. 3. A plasmid comprising the nucleicacid of claim 1 or claim
 2. 4. The plasmid of claim 3, said plasmidfurther comprising an expression control sequence capable of directingexpression of said penetrin polypeptide.
 5. A cell comprising thenucleic acid of claim 1 or claim
 2. 6. The cell of claim 5 wherein saidcell is a prokaryotic cell.
 7. The cell of claim 5 wherein said cell isa eukaryotic cell.
 8. The cell of claim 7 wherein said cell is mammaliancell.
 9. The DNA molecule identified as HB6, deposited with the ATCCunder accession number 75126.